In the last decade, small molecule tyrosine kinase inhibitors (TKIs) have revolutionized the care and prognosis of non-small cell lung cancer (NSCLC) patients whose cancers harbor oncogenic kinase alterations such as chromosomal rearrangement of anaplastic lymphoma kinase (ALK). ALK rearrangements define a unique molecular subset of NSCLC with characteristic clinicopathologic features and marked sensitivity to ALK TKIs. Based on recently conducted clinical trials, the standard treatment for advanced ALK-rearranged NSCLC consists of sequential treatment with the first-generation ALK TKI crizotinib, followed by a second-generation ALK TKI like ceritinib or alectinib. While patients derive significant clinical benefit from first-and second- generation ALK TKIs, the majority of patients relapse within a few years due to acquired resistance. Recent work has identified a number of secondary ALK resistance mutations that can mediate resistance to second- generation ALK TKIs. These on-target resistance mechanisms are found in about one-half of patients relapsing on a second-generation ALK TKI, and can be overcome in the clinic by the newest third-generation, pan- inhibitory ALK TKI lorlatinib. However, patients who initially respond to lorlatinib still relapse within a year or two due to acquired resistance. Moreover, up to one-half of patients relapsing on a second-generation ALK TKI demonstrate intrinsic resistance to lorlatinib, likely due to the development of off-target, or ALK-independent, resistance mechanisms. As treatment options are extremely limited after failure of multiple ALK TKIs, there is an urgent need to understand resistance so that new and effective therapies can be developed. In this application, we seek to elucidate molecular mechanisms underlying both acquired and intrinsic resistance to the third-generation ALK TKI lorlatinib. We will use in vitro generated models of resistance, comprehensive genetic assessment of lorlatinib-resistant patient specimens, and accelerated mutagenesis screens to identify lorlatinib-resistant ALK mutations. We will test whether these mutations are susceptible to other clinically available ALK TKIs. To define ALK-independent mechanisms of resistance, we will perform two independent screens in cell lines derived directly from patient biopsies, one involving combinations of drugs and the other utilizing CRISPR-based genome editing technology. Based on the results, we will design and test novel combinatorial strategies to overcome ALK-independent resistance in vitro and in vivo. As tumor heterogeneity may play an important role in driving the development of resistance, we will evaluate both intra- and inter- tumoral heterogeneity by sequencing single cancer cells, spatially distinct sites of disease obtained at autopsy, and circulating tumor DNA. These results will lay the groundwork for future studies investigating the impact of tumor heterogeneity on TKI response in the clinic. Overall, the proposed studies should lead to fundamental insights into the biology of TKI resistance and to the discovery of actionable mechanisms of resistance that may ultimately translate into highly effective, life-prolonging therapies for our patients.